Identification and monitoring of systemic lupus erythematosus

ABSTRACT

A method for identifying or monitoring SLE in an individual is provided. The method includes quantitating complement component C4d on the surfaces of platelets and comparing the amounts of C4d to reference levels of C4d on platelets of individuals without SLE and/or on platelets of the individual obtained at a different time. Kits for use in the above-described methods are provided along with computer readable media tangibly embodying executable instructions to perform the methods.

CROSS-REFERENCE TO RELATION APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 10/552,177,filed Oct. 5, 2005, which is a U.S. National Stage of InternationalApplication No. PCT/US04/011982, filed Apr. 16, 2004, which waspublished in English under PCT Article 21(2), which in turn claims thebenefit of U.S. Provisional Application No. 60/463,447, filed Apr. 16,2003.

FIELD OF THE INVENTION

Identification and/or monitoring of patients with systemic lupuserythematosus, including methods and kits for carrying out thisactivity.

BACKGROUND OF THE INVENTION

Systemic lupus erythematosus (SLE) or lupus is the prototypic autoimmunedisease resulting in multiorgan involvement. This anti-self response inSLE patients is characterized 10 by autoantibodies directed against avariety of nuclear and cytoplasmic cellular components. Theseautoantibodies bind to their respective antigens, forming immunecomplexes that circulate and eventually deposit in tissues. This immunecomplex deposition causes chronic inflammation and tissue damage.

Both diagnosing and monitoring disease activity are problematic inpatients with SLE. Diagnosis is problematic because the spectrum ofdisease is broad and ranges from subtle or vague symptoms tolife-threatening multi-organ failure. There also are other diseases withmulti-system involvement that can be mistaken for SLE, and vice versa.Criteria were developed for the purpose of disease classification in1971 (Cohen, A S, et al., 1971, Preliminary criteria for theclassification of systemic lupus erythematosus. Bull Rheum Dis21:643-648) and revised in 1982 (Tan, E M, et al., 1982. The 1982revised criteria for the classification of systemic lupus erythematosus.Arth Rheum 25:1271-1277) and 1997 (Hochberg, M C. 1997. Updating theAmerican College of Rheumatology revised criteria for the classificationof systemic lupus erythematosus. Arth Rheum 40: 1725). These criteriaare meant to ensure that patients from different geographic locationsare comparable. Of the 11 criteria, the presence of four or more, eitherserially or simultaneously, is sufficient for classification of apatient as having SLE. Although the criteria serve as useful remindersof those features that distinguish lupus from other related autoimmunediseases, they are

unavoidably fallible. Determining the presence or absence of thecriteria often requires interpretation. If liberal standards are appliedfor determining the presence or absence of a sign or symptom, one couldeasily diagnose a patient as having SLE when in fact they do not.Similarly, the range of clinical manifestations in SLE is much greaterthan that described by the eleven criteria and each manifestation canvary in the level of activity and severity from one patient to another.To further complicate a difficult diagnosis, symptoms of SLE continuallyevolve over the course of the disease. New symptoms in previouslyunaffected organs can develop over time. There is no definitive test forSLE and, thus, it is often misdiagnosed.

Monitoring disease activity also is problematic in caring for patientswith SLE. Lupus progresses in a series of flares, or periods of acuteillness, followed by remissions. The symptoms of a flare, which varyconsiderably between patients and even within the same patient, includemalaise, fever, symmetric joint pain, and photosensitivity (developmentof rashes after brief sun exposure). Other symptoms of lupus includehair loss, ulcers of mucous membranes and inflammation of the lining ofthe heart and lungs, which leads to chest pain. Red blood cells,platelets and white blood cells can be targeted in lupus, resulting inanemia and bleeding problems. More seriously, immune complex depositionand chronic inflammation in the blood vessels can lead to kidneyinvolvement and occasionally kidney failure, requiring dialysis orkidney transplantation. Since the blood vessel is a major target of theautoimmune response in lupus, premature strokes and heart disease arenot uncommon. Over time, however, these flares can lead to irreversibleorgan damage. In order to minimize such damage, earlier and moreaccurate detection of disease flares would not only expedite appropriatetreatment, but would reduce the frequency of unnecessary interventions.From an investigative standpoint, the ability to uniformly describe the“extent of inflammation” or activity of disease in individual organsystems or as a general measure is an invaluable research tool.Furthermore, a measure of disease activity can be used as a responsevariable in a therapeutic trial.

Two of the most commonly used instruments for SLE diagnosis are theSystemic Lupus Erythematosus Disease Activity Index (SLEDAI)(Bombardier, C., D. D. Gladman, et al. (1992). Derivation of the SLEDAI.A disease activity index for lupus patients. The Committee on PrognosisStudies in SLE. Arth Rheum 35: 63040), and the Systemic Lupus ActivityMeasure (SLAM) (Liang, M. H., S. A. Socher, et al. (1989). Reliabilityand validity of six systems for the clinical assessment of diseaseactivity in systemic lupus erythematosus. Arth Rheum 32: 1107-18). TheSLEDAI includes 24 items, representing nine organ systems. The variablesare obtained by history, physical examination and laboratory assessment.Each item is weighted from 1 to 8 based on the significance of the organinvolved. For example, mouth ulcers are scored as 2, while seizures arescored as 8. The laboratory parameters that are included in the SLEDAIinclude white blood cell count, platelet count, urinalysis, serum C3, C4and anti-dsDNA. The total maximum score is 105. The SLAM includes 32items representing 11 organ systems. The items are scored not only aspresent/absent, but graded on a scale of 1 to 3 based on severity. Thetotal possible score for the SLAM is 86. Both the SLEDAI and the SLAMhave been shown to be valid, reliable, and sensitive to change over time(Liang, M. H., S. A. Socher, et al. (1989). Reliability and validity ofsix systems for the clinical assessment of disease activity in systemiclupus erythematosus. Arth Rheum 32:1107-18), and are widely used inresearch protocols and clinical trials. These indices are particularlyuseful for examining the value of newly proposed serologic orinflammatory markers of disease activity in SLE.

Despite the obvious utility of these instruments, there are somedrawbacks. First, there is not always complete agreement between theSLAM and the SLEDAI in the same set of patients. There are severalpossible reasons for these discrepancies. Unlike the SLEDAI, the SLAMincludes constitutional symptoms such as fatigue and fever, which may ormay not be considered attributable to active SLE; this activity indexrelies on physician interpretation. In addition, the SLEDAI does notcapture mild degrees of activity in some organ systems and does not havedescriptors for several types of activity, such as hemolytic anemia. Forthese and other reasons, most studies incorporate more than one measureof disease activity. A general review of the state of the art can befound in Ramsey-Goldman, R. and Manzi, S. Systemic Lupus Erythematosus.In: Goldman and Hatch, Ed. Women and Health. Academic Press, San Diego,Calif. 2000: 704-723.

The complement system consists of a complex network of more than 30functionally linked proteins that interact in a highly regulated mannerto provide many of the effector functions of humoral immunity andinflammation, thereby serving as the major defense mechanism againstbacterial and fungal infections. This system of proteins acts againstinvasion by foreign organisms via three distinct pathways: the classicalpathway (in the presence of antibody) or the alternative pathway (in theabsence of antibody) and the lectin pathway. Once activated, theproteins within each pathway form a cascade involving sequentialself-assembly into multimolecular complexes that perform variousfunctions intended to eradicate the foreign antigens that initiated theresponse.

The classical pathway is usually triggered by an antibody bound to aforeign particle. It consists of several components that are specific tothe classical pathway and designated C1, C4, C2, (in that order in thepathway).

In the classical pathway, the first component C1q is bound to anantigen-antibody complex, activating the pathway. This event is followedby sequential activation of the two serine proteases C1 r and C1s.Activated C1s has two substrates, the final two proteins of theclassical pathway, namely C4 and C2. Protein C4 is cleaved into C4a andC4b. Protein C2 is cleaved to form C2a and C2b. Fragments C4b and C2aassemble to form C4b2a, which cleaves protein C3 into C3a and C3b, whichcompletes activation of the classical pathway.

Fragments C4b and C3b are subject to further degradation by Factor 1.This factor cleaves C4b to generate C4d and also cleaves C3b, togenerate iC3b followed by C3d. Thus, activation of the classical pathwayof complement can lead to deposition of a number of fragments, includingC4d and iC3b on immune complexes or other activating surfaces. Thesefragments are ligands for complement receptor type 1 (CR1) onerythrocytes or red blood cells.

BRIEF SUMMARY OF THE INVENTION

It has been found that increased levels of complement C4d are present onthe surfaces of platelets of individuals with systemic lupuserythematosis as compared to platelets obtained from healthyindividuals. As shown herein, high levels of complement C4d associatedwith platelets correlates strongly with the presence of systemic lupuserythematosis in an individual and can be used for diagnosis andmonitoring of systemic lupus erythematosis in the individual. Theinvention includes determining (quantitating) levels of complement C4dassociated with platelets obtained from patients in which systemic lupuserythematosis is suspected or is present, and comparing those levels tothose of platelets obtained from healthy individuals. In one embodiment,the amount of C4d associated with platelets is measured by detection offluorescence by flow cytometry.

In one aspect, a method is provided for identifying systemic lupuserythematosus in an individual, comprising quantitating complement C4dassociated with platelets obtained from the individual, and comparingthe quantity of complement C4d associated with platelets obtained fromthe individual with quantities of complement C4d associated withplatelets obtained from individuals not having systemic lupuserythematosus, wherein greater quantities of C4d associated withplatelets obtained from the individual as compared to quantities of C4dassociated with platelets obtained from individuals not having systemiclupus erythematosus correlates with the presence of systemic lupuserythematosus in the patient.

In another aspect, a method is provided for monitoring disease activityof systemic lupus erythematosus in an individual, comprisingquantitating complement C4d associated with platelets obtained from theindividual and comparing the quantity of complement C4d associated withplatelets obtained from the individual with quantities of complement C4dassociated with platelets obtained from either the individual at one ormore different times or individuals not having systemic lupuserythematosus, wherein greater quantities of C4d associated withplatelets obtained from the individual as compared to quantities of C4dassociated with platelets obtained from either the individual at one ormore different times or individuals not having systemic lupuserythematosus correlates with the presence of active systemic lupuserythematosus in the patient.

In still another aspect, a method is provided comprising quantitatingcomplement C4d associated with platelets obtained from the individualand comparing the quantity of complement C4d associated with plateletsobtained from the individual with quantities of complement C4dassociated with platelets obtained from either the individual at one ormore different times or individuals not having systemic lupuserythematosus, wherein greater quantities of C4d associated withplatelets obtained from the individual as compared to quantities of C4dassociated with platelets obtained from either the individual at one ormore different times or individuals not having systemic lupuserythematosus correlates with the presence of systemic lupuserythematosus in the patient.

In another embodiment a kit for use in identifying systemic lupuserythematosus in an individual, comprising a package containing aconjugate of an antibody specific for complement C4d with a firstlabeled moiety and instructions relating to use of the conjugate toidentify complement C4d associated with platelets.

In yet another embodiment, a computer readable medium is provided,tangibly embodying executable instructions to perform a method, themethod comprising: receiving data corresponding to a determination ofcomplement component C4d deposited on surfaces of platelets; retrievinga reference value for complement component C4d deposited on surfaces ofplatelets of individuals; and comparing the data with the referencevalue.

Also provided is a computer readable medium tangibly embodyingexecutable instructions to perform a method for identifying systemiclupus erythematosus in an individual, the method comprising:quantitating complement C4d associated with platelets obtained from theindividual; and comparing the quantity of complement C4d associated withplatelets obtained from the individual with quantities of complement C4dassociated with platelets obtained from individuals not having systemiclupus erythematosus, wherein greater quantities of C4d associated withplatelets obtained from the individual as compared to quantities of C4dassociated with platelets obtained from individuals not having systemiclupus erythematosus correlates with the presence of systemic lupuserythematosus in the patient.

DETAILED DESCRIPTION OF THE INVENTION

The methods of this invention enable the identification and/ormonitoring of SLE. Because this condition is a serious health problem,there is a need for relatively accurate and early diagnosis of thiscondition. Likewise, the ability to monitor the activity of this diseaseis of great importance. In the most general sense, the methods of thisinvention are based on the discovery by the inventors that adetermination of C4d deposited on surfaces of platelets of a patient canserve as a diagnostic marker for SLE.

In diagnosing the occurrence, or previous occurrence, of SLE, complementcomponent C4d deposited on surfaces of platelets in a sample isquantitated. The amount of C4d on the surfaces of the platelets is thencompared with the quantities of C4d usually found on the surfaces ofplatelets of individuals not having SLE. For accuracy, the methodsdescribed herein measure C4d “associated with” platelets. The reason forthis distinction arises form the fact that some methods, such as flowcytometry, measure C4d on the surface of intact platelets directly,while other methods quantitate surface C4d indirectly in a sample ofdisrupted platelets, and do not literally measure C4d on the surfaces ofplatelets. As such, as used herein, C4d is said to be “associated with”platelets, which means either C4d is on the surface of a platelet or itis present in a disrupted platelet sample, but in any case is either adirect or indirect measurement of C4d deposited on the surfaces ofplatelets.

In monitoring disease activity in a patient with SLE, the samedetermination is made in the patient's blood sample and is then comparedwith determinations of the quantities of C4d associated with plateletsin a sample obtained from the same patient in the past.

In general, samples of blood are obtained from the patient and aretreated with EDTA (ethylenediaminetetraacetate) to inhibit complementactivation. The samples can be maintained at room temperature or undercold conditions. Assays preferably are run within 48 hours.

Complement C4d can be quantitated by a number of methods, including flowcytometry, ELISA using platelet lysates, and radioimmunoassay (RIA). Inone embodiment, C4d is quantitated by flow cytometric methods, withmeasurements taken by direct or indirect immunofluorescence usingpolyclonal or monoclonal antibodies specific for C4d. Typically, themean fluorescence channel (MFC) for platelet C4d is determined. Flowcytometry also may be used for monitoring disease activity in patientsknown to have SLE. In this method, anti-C4d antibody conjugated with afirst fluorescent moiety is added to a blood sample. Preferably aplatelet-specific antibody, conjugated with a second fluorescent moietythat is detectably different from the first fluorescent moiety, also isadded to the blood sample to facilitate quantitation by cell sorting ofC4d deposited on platelets and not on other blood components.

Kits for use in conducting flow cytometric assays for both thediagnosing of disease and monitoring of disease activity are alsoprovided. Kits may include, in a package, any of the various reagentsneeded to perform the methods described herein. For example, forimmunofluorescence assays, the kits generally will comprise a conjugateof an antibody specific for complement component C4d with a labeledmoiety, such as a fluorescent moiety, and preferably also a conjugate ofa platelet-specific antibody with a different fluorescent moiety, forexample and without limitation, a monoclonal antibody specific forplatelet CD42 b. The antibody or antibodies typically are monoclonal.Additionally, the kits will comprise such other material as may beneeded in carrying out assays of this type, for example and withoutlimitation: buffers; radiolabelled antibodies, secondary antibodies, asin the case of ELISA or RIA assays, that are conjugated with a labelingmoiety or other tag, such as avidin or biotin, further conjugated with afluorochrome or enzyme such as alkaline phosphatase or peroxidase;calorimeter reagents and instructions for use of the anti-C4d antibodyand/or other components of the kit, to quantitate C4d levels onplatelets.

Antibodies useful in the described methods and kits are known.Hybridomas secreting Anti-CD42b antibodies are available, for exampleand without limitation, from Becton Dickinson Immunocytometry Systems,San Jose, Calif. Anti-C4d antibodies are available from Quidel Corp. inSan Diego, Calif. (#A213) and are generally described in Rogers, J., N.Cooper, et al. Complement activation by beta-amyloid in Alzheimer'sdisease. PNAS 89:10016-10020, 1992; Schwab, C., et al. Neurofibrillarytangles of Guam Parkinson-dementia are associated with reactivemicroglia and complement proteins. Brain Res 707(2):196 1996; Gemmell,C. A flow cytometric immunoassay to quantify adsorption of complementactivation products on artificial surfaces. J Biomed Mater Res37:474-480, 1997; and, Stoltzner, S. E., et al. Temporal accrual ofcomplement proteins in amyloid plaques in patients with Down's syndromewith Alzheimer's disease. Am J Path 156:489-499, 2000. Binding reagentsspecific to C4d and/or CD42b, including, without limitation, FvFragments, single chain Fv Fragments, Fab fragments, F(ab)2 fragments ormultivalent binding reagents, are understood as synonymous with“antibodies” or “antibody”.

The determination of the C4d and CD42b values alternatively may beconducted using a number of standard measurement techniques such asELISA or RIA. Instead of fluorescent labels, used for flow cytometry,there may be used labels of other types, such as radioactive andcolorimetric labels. If such other types of assays are to be used, thekits will comprise antibodies, typically monoclonal antibodies, specificfor C4d and CD42b conjugated with appropriate labels such as radioactiveiodine, avidin, biotin or enzymes such as alkaline phosphatase orperoxidase.

Diagnosis of a patient with SLE is carried out by comparing thedetermination of C4d with a base value or range of values for thequantities of C4d typically present on the surfaces of platelets innormal individuals. In normal individuals, C4d is not present atdetectable levels. When using flow cytometric measurement with indirectimmunofluorescence, the MFC of C4d on platelets of healthy individualsranges from −1.17 to 0.87 (mean −0.39). (Table I and Table IV). The MFCof platelet C4d in patients having SLE was higher than that of healthyindividuals and ranged from −0.85 to 31.67 (mean 2.65). (Table II andTable IV).

A particular feature of the methods of this invention is to indicate orreflect SLE activity that has occurred in the patient during thepreceding several weeks or even several months. It is possible, usingthis procedure, to identify the occurrence of a flare-up of SLE duringthe previous few weeks or possibly even the previous several months dueto persistence of C4d deposited on the surface of platelets.

Automation and Computer Software

The determinations of C4d and the diagnostic and disease activitymonitoring methods described above can be carried out manually, butoften are conveniently carried out using an automated system and/orequipment in which the blood sample is analyzed automatically to makethe necessary determination or determinations, and the comparison withthe base or reference value is carried out automatically, using computersoftware appropriate to that purpose.

Thus, in one aspect, the invention comprises a method for diagnosing ormonitoring systemic lupus erythematosus in an individual comprising (a)automatically determining, in a blood sample from the individualcontaining platelets, complement component C4d deposited on surfaces ofplatelets in the sample, and (b) automatically comparing saiddeterminations with reference values for component C4d on surfaces ofplatelets.

In reference to FIG. 1, one embodiment of the present inventioncomprises a system 100, which may include a device 102 operating underthe command of a controller 104. The broken lines are intended toindicate that in some implementations, the controller 104, or portionsthereof considered collectively, may instruct one or more elements ofthe device 102 to operate as described. Accordingly, the functionsdescribed herein may be implemented as software executing in the system100 and controlling one or more elements thereof. An example of a device102 in accordance with one embodiment of the present invention is ageneral-purpose computer capable of responding to and executinginstructions in a defined manner. Other examples include aspecial-purpose computer including, for example, a personal computer(PC), a workstation, a server, a laptop computer, a web-enabledtelephone, a web-enabled personal digital assistant (PDA), amicroprocessor, an integrated circuit, an application-specificintegrated circuit, a microprocessor, a microcontroller, a networkserver, a Java virtual machine, a logic array, a programmable logicarray, a micro-computer, a mini-computer, or a large frame computer, orany other component, machine, tool, equipment, or some combinationthereof capable of responding to and executing instructions.Furthermore, the system 100 may include a central processing engineincluding a baseline processor, memory, and communications capabilities.The system 100 also may include a communications system bus to enablemultiple processors to communicate with each other. In addition, thesystem 100 may include a storage 106 in the form of a disk drive,cartridge drive, and control elements for loading new software._Inembodiments of the invention, one or more reference values may be storedin a memory associated with the device 102. After data corresponding toa determination of complement C4d is obtained (e.g., from an appropriateanalytical instrument), the device 102 may compare the C4d data with oneor more appropriate reference values. After this comparison takes place,the device 102 can automatically determine if the data corresponding tothe determination of complement C4d is associated with SLE.

Embodiments of the controller 104 may include, for example, a program,code, a set of instructions, or some combination thereof, executable bythe device 102 for independently or collectively instructing the device102 to interact and operate as programmed. One example of a controller104 is a software application (e.g., operating system, browserapplication, client application, server application, proxy application,on-line service provider application, and/or private networkapplication) installed on the device 102 for directing execution ofinstructions. In one embodiment, the controller 104 may be a Windows™,based operating system. The controller 104 may be implemented byutilizing any suitable computer language (e.g., C\C++, UNIX SHELLSCRIPT, PERL, JAVA, JAVASCRIPT, HTML/DHTML/XML, FLASH, WINDOWS NT,UNIX/LINUX, APACHE, RDBMS including ORACLE, INFORMIX, and MySQL) and/orobject-oriented techniques.

In one embodiment, the controller 104 may be embodied permanently ortemporarily in any type of machine, component, physical or virtualequipment, storage medium, or propagated signal capable of deliveringinstructions to the device 102. In particular, the controller 104 (e.g.,software application and/or computer program) may be stored on anysuitable computer readable media (e.g., disk, device, or propagatedsignal), readable by the device 102, such that if the device 102 readsthe storage medium, the functions described herein are performed. Forexample, in one embodiment, the controller 104 may be embodied invarious computer-readable media for performing the function of:

-   -   (a) receiving data corresponding to a determination of        complement component C4d deposited on surfaces of platelets;    -   (b) retrieving a reference value for complement component C4d        deposited on surfaces of platelets of individuals; and    -   (c) comparing the data in (a) with the reference value of (b).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

EXAMPLES AND EXPERIMENTAL DATA

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters that could be changed or modified to yieldessentially similar results.

Example 1 Assays of Platelet C4d in Healthy Controls: Negative

Twenty-five healthy individuals were studied. As shown in Table I, C4dwas not detected on platelets of each of the twenty-five healthyindividuals. Samples of 1 mL of EDTA-anticoagulated peripheral bloodwere taken from each individual and used as a source of platelets. Theplatelets were washed and resuspended in Fluorescence-Activated CellSorter (FACS) buffer. Levels of C4d and CD42b were measured by two colorindirect immunofluorescence using monoclonal antibodies specific for C4dand CD42b, respectively. Levels of C4d and CD42b are quantitated by Flowcytometry using a FACSCalibur cytometer (Becton Dickinson). Theplatelets were identified by forward and side scatter andCD42b-fluorescence, and the mean fluorescence channel (MFC) wasdetermined for C4d.

More particularly, blood was drawn into 4 cc Vacutainer tubes containing7.2 mg EDTA as an anticoagulant (Becton Dickinson, Franklin Lakes,N.J.), and processed within two hours. Whole blood was diluted 1/10 inphosphate buffered saline (PBS). 10 μl aliquots of the diluted bloodwere immufluorescently labeled for flow cytometry with 0.25 μg ofPE-labeled anti-CD42b monoclonal antibody (Becton DickinsonImmunocytometry Systems, San Jose, Calif.) to identify platelets, and0.25 μg of one of the following monoclonal antibodies conjugated toAlexa Fluor 488 (Molecular Probes, Eugene, Oreg.): anti-C4d (QuidelCorp., San Diego, Calif.) or the isotype control MOPC21. Samples wereincubated 10 min at room temperature, then diluted with 0.5 ml cold PBSand analyzed on a FACSCalibur flow cytometer (Becton DickinsonImmunocytometry Systems, San Jose, Calif.). Platelets wereelectronically gated by forward scatter properties and expression ofCD42b, a platelet-specific marker. Nonspecific binding ofimmunoglobulins to platelets was determined by performing identicalassays in parallel using the isotype control antibody MOPC21 (obtainedfrom American Type Culture Collection, Manassas, Va.). Specific bindingof anti-C4d and anti-CD42b were determined by subtracting the MFCobtained with MOPC21 from the MFC obtained with anti-C4d and anti-CD42b,respectively.

Example 2 Assays of Platelet C4d to Distinguish Patients with SLE fromHealthy Controls

This example describes conducting assays on patients to diagnosesystemic lupus erythematosus, and to establish reference values orranges of values for complement component C4d.

For this purpose, we recruited 115 patients with lupus from ouroutpatient office. A single determination of platelet C4d was made in115 individuals who met ACR criteria for the diagnosis of SLE (Table II)and in 35 healthy controls (Table I). The mean and median values of CR1and C4d for patients with SLE and healthy controls are shown in TableIV. Whereas the mean value for C4d in healthy individuals was −0.39, themean value for C4d among patients with SLE was 2.65 (p=0.0001).

TABLE I Healthy Controls (n = 25) Mean MFC = −0.39 Range (−1.17)-(+0.87)Platelet C4d MFC 2003 −0.28 2005 −0.23 2006 −0.51 2007 −0.05 2008 0.202009 0.15 2010 −0.39 2011 −0.71 2013 −0.96 2017 0.87 2020 −0.29 2021−0.56 2022 0.38 2025 −0.73 2026 −0.24 2027 −0.34 2028 −0.74 2029 −0.052030 −0.51 2031 −1.03 2032 −0.42 2034 −0.71 2035 −0.86 2036 −0.48 2037−1.17

TABLE II SLE Patients (n = 115) MEAN = 2.65 RANGE = (−0.85)-(+31.67)Patient Platelet ID C4d 1001 3.79 1002 0.59 1003 1.36 1006 4.64 10080.00 1009 4.58 1010 20.82 1011 0.74 1012 1.47 1013 10.37 1014 −0.40 10159.44 1016 4.18 1017 1.05 1018 1.37 1019 0.00 1021 0.92 1027 0.00 10302.36 1031 2.76 1032 0.38 1034 0.00 1035 0.14 1036 7.11 1037 0.00 10380.70 1039 12.60 1043 0.00 1044 0.94 1045 0.00 1046 0.40 1047 1.52 10480.00 1050 3.80 1052 5.92 1053 1.11 1055 1.24 1056 14.90 1057 0.00 10590.03 1060 0.00 1061 4.41 1063 0.84 1064 −0.15 1066 31.67 1067 0.18 10682.63 1071 0.03 1072 0.00 1073 1.61 1074 0.02 1075 23.61 1076 8.60 1078−0.17 1079 1.61 1080 2.59 1081 6.59 1082 2.52 1083 0.05 1084 0.06 10854.48 1086 −0.06 1087 0.58 1089 −0.10 1090 9.71 1091 −0.14 1092 −0.381093 0.34 1094 −0.53 1095 −0.11 1096 −0.85 1097 −0.12 1098 −0.24 1099−0.31 1100 0.52 1101 1.49 1102 8.74 1103 −0.35 1104 −0.22 1105 −0.311106 0.90 1108 0.89 1109 1.06 1110 2.69 1111 0.59 1112 1.53 1113 1.031115 0.92 1116 2.16 1117 1.26 1118 4.97 1119 0.62 1120 0.94 1121 6.171122 3.75 1123 1.78 1124 1.20 1125 3.10 1126 2.57 1127 1.47 1128 1.671129 1.29 1130 6.35 1131 2.36 1132 7.25 1134 1.47 1135 0.86 1136 1.701137 2.70 1138 0.92 1139 1.30 1140 1.34 1141 1.09 1142 3.82 1143 1.60

In SLE patients compared to healthy controls, the sensitivity andspecificity of these measures were 39% and 100%, respectively (Table V).

Example 3 Assay of Platelet C4d for Distinguishing Patients with SLEfrom Patients with Other Diseases

These studies of patients with SLE vs. healthy controls were followed bystudies to compare patients with SLE with patients diagnosed withdiseases other than SLE (n=103). For this comparison, we studiedpatients with systemic sclerosis (n=13), rheumatoid arthritis (n=17),osteoarthritis (n=2), hepatitis C virus infection (n=14),polymyositis/dermatomyositis (n=18), Sjogren's syndrome (n=2),urticarial vasculitis (n=1), sickle cell anemia (n=8), overlapsyndrome/undifferentiated connective tissue disease (n=12),leukemia/lymphoma (n=9), primary Raynaud's syndrome (n=3), hemophilia(n=2), and psoriatic arthritis (n=2). A single determination of plateletC4d was made using the same assay. The mean and median values of C4d forpatients with SLE, as compared with patients with other diseases, areshown in Table IV. Whereas the mean value for C4d in patients with otherdiseases was 0.53, the mean value for C4d among patients with SLE was2.59 (p=0.0001). In SLE patients compared to patients with otherdiseases, the sensitivity and specificity of these measures were 39% and94%, respectively (Table V).

TABLE III Other Diseases (n = 103) MEAN = 0.68 RANGE = (−3.29)-(+10.22)Patient Platelet ID C4d 3003 1.11 3004 0.42 3014 0.66 3021 0.17 30220.28 3023 0.00 3028 0.56 3029 0.53 3030 1.03 3031 10.22 3034 1.02 30351.15 3036 1.33 4001 0.08 4002 0.73 4008 0.91 4011 1.07 4018 0.02 40191.04 4020 0.20 4021 0.44 4022 0.10 4023 1.17 4024 0.03 4025 0.28 40260.32 4027 0.06 4028 1.22 4030 0.53 4033 1.03 4034 1.15 5001 1.05 50030.55 6001 2.12 6002 1.04 6003 0.47 6004 0.50 6005 0.04 6006 0.40 60070.04 6008 0.18 6009 0.11 6010 3.58 6011 0.14 6012 0.40 6013 0.71 60140.21 6015 1.28 6016 1.01 6017 2.00 8001 0.54 8002 1.46 8003 0.05 80041.39 8005 1.83 8007 0.03 8008 0.02 8009 0.10 8012 0.29 8013 0.34 80150.56 8018 0.24 8019 0.05 8020 0.25 10001 0.89 12001 0.29 12002 0.0612003 0.16 12004 0.46 12005 0.33 12006 0.05 12007 0.14 12008 0.10 130031.37 13007 0.46 13008 0.08 13009 0.47 13010 0.13 13011 1.45 13012 0.1513013 0.42 13014 0.20 13015 1.04 13016 0.97 13017 0.58 14001 1.15 140021.63 14003 0.52 14004 0.67 14005 1.25 14006 1.47 14007 0.17 14008 0.1414009 0.05 15002 0.11 15003 0.40 15004 0.89 16001 1.06 16002 0.14 17001−3.29 17002 0.17 18001 0.80 18002 1.00

Example 4 Assays of CR1 and C4d for Measuring Disease Activity inPatients with SLE

We then examined the utility of platelet C4d levels in measuring diseaseactivity as defined by the Systemic Lupus Erythematosus Disease ActivityIndex (SLEDAI). We present the results of the first 115 lupus patientsentered into the study. Using a univariate linear regression model,platelet C4d was the most significant predictor of disease activity(P=0.002).

TABLE IV Analysis of Platelet C4d Platelet-C4d Mean Standard DeviationMedian SLE (n = 15) 2.65 4.76 2.13 Healthy Controls (n = 25) −0.39 0.46−0.42 Other Diseases  (n = 103) 0.68 1.18 0.46 Comparison C4 (p values)SLE vs. Healthy Controls 0.0001 SLE vs. Other Diseases 0.0001

TABLE V Analysis of Platelet C4d Comparison Sensitivity Specificity SLEvs. Healthy Control .39 1.00 SLE vs. Other Diseases .39 .94

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the techniques of this invention that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims.

1. A computer readable medium, storing computer-executable instructionsfor implementing an evaluation tool using an automated system, whereinthe automated system comprises memory and a processor, and wherein theevaluation tool evaluates data corresponding to a determination ofcomplement component C4d associated with platelets, thecomputer-executable instructions causing the processor to: receive datacorresponding to the complement component C4d associated with plateletsin a platelet sample from an individual; store the data corresponding tocomplement component C4d in the memory of the automated system; store aretrieved a reference value for complement component C4d associated withplatelets of individuals; and compare the received data with thereference value.
 2. The computer readable medium of claim 1, thecomputer-executable instructions further causing the processor to:determine if the individual has systemic lupus erythematosus based onthe comparison of the received data and the reference value; and presentthe results of the determination.
 3. The computer readable medium ofclaim 1, the computer-executable instructions further causing theprocessor to: receive data corresponding to the complement componentC42b associated with platelets in a platelet sample from an individual;store the data corresponding to complement component C42b in the memoryof the automated system; store a retrieved a reference value forcomplement component C42b associated with platelets of individuals; andcompare the received data with the reference value for complementcomponent C42b.
 4. The computer readable medium of claim 3, thecomputer-executable instructions further causing the processor to:determine if the individual has systemic lupus erythematosus based onthe comparison of the received data and the reference value; and presentthe results of the determination.
 5. A computer readable medium tangiblyembodying executable instructions to perform a method for identifyingsystemic lupus erythematosus in an individual, the method comprising:quantitating complement C4d associated with platelets obtained from aplatelet sample from the individual; and comparing the quantity ofcomplement C4d associated with platelets obtained from the individualwith quantities of complement C4d associated with platelets obtainedfrom individuals not having systemic lupus erythematosus, wherein thequantity of C4d associated with platelets obtained from the plateletsample of the individual being higher as compared to quantities of C4dassociated with platelets obtained from platelet samples of individualsnot having systemic lupus erythematosus correlates with the presence ofsystemic lupus erythematosus in the patient.
 6. The computer readablemedium of claim 5, further comprising executable instructions forquantitating CD42b associated with platelets obtained from theindividual.
 7. The computer readable medium of claim 5, furthercomprising executable instructions for conducting a quantitation ofcomplement C4d by binding the C4d to a conjugate of an antibody specificfor complement C4d with a first labeled moiety and quantitating thefirst labeled moiety.
 8. The computer readable medium of claim 7,wherein the first labeled moiety is a fluorescent moiety.
 9. Thecomputer readable medium of claim 7, wherein the first fluorescentmoiety is quantitated by flow cytometric analysis.
 10. The computerreadable medium of claim 7, further comprising executable instructionsfor: binding CD42b to a conjugate of an antibody specific for CD42b witha second labeled moiety that is detectably different from the firstlabeled moiety; and quantitating the second labeled moiety.
 11. Thecomputer readable medium of claim 10, wherein the second labeled moietyis a fluorescent moiety.
 12. The computer readable medium of claim 11,wherein the second fluorescent moiety is quantitated by flow cytometricanalysis.
 13. A computer readable medium tangibly embodying executableinstructions to perform a method for monitoring disease activity ofsystemic lupus erythematosus in an individual, the method comprising:quantitating complement C4d associated with platelets in a plateletsample obtained from the individual; and comparing the quantity ofcomplement C4d associated with platelets obtained from the plateletsample from the individual with quantities of complement C4d associatedwith platelets obtained from either platelet samples from the individualat one or more different times or platelet samples from individuals nothaving systemic lupus erythematosus; wherein quantities of C4dassociated with platelets obtained from the individual being higher ascompared to quantities of C4d associated with platelets obtained fromeither platelet samples from the individual at one or more differenttimes or platelet samples from individuals not having systemic lupuserythematosus correlates with the presence of systemic lupuserythematosus in the patient.
 14. The computer readable medium of claim13, further comprising executable instructions for quantitating CD42bassociated with platelets obtained from the individual.